Gestational hyperglycemia can result in acute and chronic adverse outcomes for mother and offspring. In a recent genetic study of the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) cohort, PI Layden, Co- investigator Reddy, and co-authors reported a novel loci associated with maternal glycemic traits specifically during pregnancy. The lead candidate gene in the associated region encodes the hexokinase-domain containing 1 (HKDC1) protein. Therefore, the overall objective of this proposal is to understand the role of the novel gene HKDC1 in gestational glucose homeostasis. Recently, PI Layden and co-Investigator Reddy published the first characterization of HKDC1, revealing unexpectedly that HKDC1 is a fifth vertebrate hexokinase (Guo et al., 2015 Nature Communications). The goal of this proposal, which builds upon those initial findings, is to test the central hypothesis that HKDC1 is a novel hexokinase that controls glucose homeostasis during pregnancy. Testing that hypothesis is important because it will reveal new mechanisms contributing to one of the most common complications of pregnancy worldwide. The results will also have broad-reaching impacts on understanding vertebrate metabolism because hexokinase is the first step in glycolysis, and the first characterization of the fifth human hexokinase will substantially revise our basic understanding of the enzymes controlling glucose homeostasis during health and disease. The central hypothesis will be tested at three complementary levels. First, following up on the initial discovery that HKDC1 is a hexokinase, the enzymatic activity of the protein will be systematically characterized. Doing so will reveal whether HKDC1 is more likely responsible for glucose sensing or glucose metabolism, and will relate HKDC1 to the other hexokinases. Those insights are essential for understanding the role of HKDC1 in maternal health. Second, the in vivo role of HKDC1 in glucose homeostasis during pregnancy will be assessed. Tissue expression patterns suggest that HKDC1 regulates glucose homeostasis during pregnancy specifically through its role in pancreatic ? cells and liver. Through global and tissue-specific ablation mouse models, glucose homeostasis during pregnancy will be assessed. These findings will be translated to in vitro studies in human islets and primary human hepatocytes. Third, the role of HKDC1 in regulating glucose during human pregnancy will be investigated by testing the hypothesis that gestational hyperglycemia-associated genetic variants alter the regulation of HKDC1 during pregnancy. That hypothesis will be tested using novel high- throughput reporter assays (i) to discover the primary and secondary hormone and glucose response elements that regulate HKDC1 expression and (ii) to identify genetic variants that alter regulatory element function in the original GWAS population. The outcome of this proposal will be a comprehensive understanding of the activity and transcriptional regulation of HKDC1 during pregnancy. Long term, those findings will support downstream efforts to predict, prevent, and treat gestational hyperglycemia.